Functional and molecular characterization of single, (4‐hydroxy‐3‐nitrophenyl)acetyl (NP)‐specific, IgG₁⁺ B cells from antibody‐secreting and memory B cell pathways in the C57BL/6 immune response to NP

Abstract

We have used multiparameter flow cytometry to identify a population of IgG1+ IgM- antigen-specific B cells which emerges in spleens of C57BL/6 mice following immunization with the hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP). Characterization of the specificities of IgG1 antibodies produced by single, sorted IgG1+ NP+ cells in both Elispot assays and in microcultures containing lipopolysaccharide, interleukin (IL)-2, IL-4 and IL-5 indicates that the splenic IgG1+ NP+ B cell population includes both IgG1 anti-NP antibody-secreting cells and non-secreting, IgG1+ memory B cells. Each functionally discrete population of IgG1+ B cells expresses a distinctive surface phenotype defined by a wide range of B cell markers. In particular, antibody-secreting, IgG1+ cells were uniquely identified by co-expression of the matrix receptor, syndecan. The NP-specific B cell population emerging in the day 7 primary response was assessed for clonotypic diversity by amplification and direct sequencing of the rearranged V186.2 heavy chain variable region gene expressed by single, ex vivo IgG1+ NP+ lambda+ B cells. Memory B cell clones, distinguished by junctional diversity, carried either no mutation or a single mutation within rearranged V186.2, suggesting isolation of these cells at the onset of the hypermutation mechanism. This novel approach, therefore, allows the direct and unambiguous identification and characterization of individual B cell clonotypes during their initial selection and activation in antibody responses in vivo.

A genetic lesion that arrests plasma cell homing to the bone marrow

“…Phenotypic markers can be used to distinguish PCs from naïve and GC B cells. Expression of CD138 is observed on differentiating PCs, but not on other mature B cells (27)(28)(29)(30), and is accompanied by reduced levels of B220 expression (29,(31)(32)(33). By using three-color laser scanning confocal microscopy, we can identify PCs as CD138 ϩ (Fig.…”

IL-21 Receptor Is Critical for the Development of Memory B Cell Responses

“…NP-specific ELISAs were done as previously described (25,26). Briefly, for IgM and IgG ELISAs, plates were coated overnight with 20 mg/ml NP(18)-BSA (Biosearch Technologies).…”

“…NP-specific Ab-secreting cells (ASCs) were measured by ELISPOTs as previously described (25,27). Briefly, Multiscreen HA plates (Millipore, Bedford, MA) were coated with 100 ml (20 mg/ml) NP(18)-BSA (Biosearch Technologies) overnight at 4˚C and blocked the next day with RPMI 1640 plus 10% FBS.…”

Role of the Polymeric Ig Receptor in Mucosal B Cell Homeostasis

“…Alum-precipitated (4-hydroxy-3-nitrophenyl) acetyl (NP) conjugated to keyhole limpet hemocyanin (KLH; NP:KLH conjugation ratio, 17:1) was prepared as described previously (24) and was supplied by Dr. D. Tarlinton (WEHI). To generate systemic Ab responses to NP, mice were immunized with 100 g of NP 17 -KLH in 100 l of PBS by i.p.…”